Zinc finger is one of DNA-binding motives, like the helix-turn-helix motif and the leucine zipper motif. It has two cysteine residues in the amino terminus region and two histidine residues in the carboxyl terminus region, and takes a three dimensional structure in which zinc (Zn) coordinates with these residues. Since zinc finger has an extremely strong DNA-binding ability, artificial DNA-binding proteins that utilize the motif to strongly bind to DNA (henceforth also referred to as “AZP” in this specification) have been proposed, and there have been reported AZPs designed so that they can recognize a specific nucleotide sequence by using the nondegenerate recognition code table (Japanese Patent Unexamined Publication (KOHYO) No. 2004-519211; Biochemistry, 41, pp. 7074-7081, 2002).
One zinc finger motif can recognize 3 or 4 bps and bind to the base pairs, and by connecting zinc fingers with a peptide linker, length of nucleotide sequence desired to be specifically bound by zinc fingers can be controlled. The fourth recognition nucleotide sequence of the zinc finger motif is an antisense strand, and overlaps with the first recognition nucleotide sequence of the following zinc finger motif, and therefore, N zinc finger motifs recognize a nucleotide sequence of 3N+1 bps, and bind thereto (see FIG. 1).
It has been reported that infection of plant DNA viruses can be controlled by using the AZP (J. Virology, 79, pp. 2614-2619, 2005). This publication reports infection-controlling effect of AZP for the plant DNA virus, beet severe curly top virus (BSCTV), in Arabidopsis thaliana. In this method, a means of inhibition by AZP is applied against the binding of a replication protein (Rep) to the Rep binding site (direct repeats) on the replication origin, which binding is required for the start of virus replication, and this method includes design of AZP to inhibit virus replication so that the AZP has a DNA-binding ability higher than that of Rep on the basis of the direct repeats of the replication origin. However, since the replication origin has a virus-specific nucleotide sequence, this method, including the block of the direct repeats of Rep by the AZP, has a problem that use of each different AZP is required against each of various plant viruses. From this point of view, it is desired to provide a means for achieving infection-controlling effect for various plant viruses with a single AZP.
A disease causing dwarf, mottle-leaf, yellowing and reduction of heading in wheat was found in Hanzhong, Shaanxi Province of the People's Republic of China, and the wheat dwarf virus (WDV, this virus may be henceforth referred to with the abbreviation, WDV) was identified as the causative virus of the disease (Zhiwu Baohu (ISSN: 0529-1542), Vol. 34, No. 2, pp. 17-21, 2008). It was also revealed that the genomic structures of several kinds of WDVs isolated in Hanzhong were the same, and they belonged to the genus Mastrevirus of the family Geminiviridae. Concerning WDV, there are also reports of Plant Pathology, 57, pp. 838-841, 2008; Plant Pathology, 58, pp. 1161-1169, 2009, as well as Virus Genes, 34, pp. 359-366, 2007, and the like.
Geminivirus is a generic name of viruses having one or two single-stranded cyclic DNAs that infect plants, and geminiviruses include various plant viruses, and roughly classified into four kinds, those belonging to the genera Begomovirus, Topocuvirus, Curtovirus, and Mastrevirus. Examples of the viruses belonging to the genus Begomovirus include, for example, tomato yellow leaf curl virus (TYLCV), potato yellow mosaic virus (PYMV), bean golden mosaic virus (BGMV), and the like. Examples of the viruses belonging to the genus Mastrevirus include, in addition to WDM mentioned above, maize streak virus (MSV), miscanthus streak virus (MiSV), tobacco yellow dwarf virus (TYDV), chloris straite mosaic virus (CSMV), and the like. Examples of the viruses belonging to the genus Topocuvirus include tomato pseudo-curly top virus (TPCTV), and examples of the viruses belonging to the genus Curtovirus include beet mild curly top virus (BMCTV) (refer to FIG. 3).
When a geminivirus enters into a plant, it first becomes a double-stranded cyclic DNA by utilizing an endogenous factor of the plant. Then, the replication protein (Rep) derived from the virus binds to the Rep-binding site locating upstream of a stem loop of intergenic region (IR). Rep is a multi-functional protein, and it binds to the Rep-binding site, introduces a nick into a nine-nucleotide sequence of the loop moiety of the stem loop, and covalently binds to the 5′ end of the DNA introduced with the nick. Then, DNA synthesis is started from the 3′ end by using one of the strands as the template, and when one copy of the genome is synthesized, a nick is introduced into the newly formed nine-nucleotide sequence by Rep. The DNA corresponding to one copy of the genome simultaneously excised is ligated by Rep, thus the single-stranded cyclic DNA is replicated, and Rep covalently binds to the newly formed 5′ end. Replication of geminivirus is attained by repetition of this process, and all the materials required for the replication other than Rep are derived from the plant (refer to FIG. 2 as well as Kagaku to Seibutsu (Bioscience & Biotechnology), 41, pp. 311-317, 2003, and the like).
It is known that Rep cleaves only a single-stranded DNA, and in order for Rep to cleave a viral DNA, it is necessary that the viral DNA forms a stem loop structure. It is known that a nucleotide sequence that forms such stem loop is very highly conserved in viruses belonging to the genus Begomovirus among the geminiviruses. In general, the stem region consists of nine GC pairs and two AT pairs, and the loop region consists of 11 or 12 nucleotides, and comprises TT, TTT, TA, or ATA, followed by a nucleotide sequence of TAATATTAC (refer to Kagaku to Seibutsu, 41, pp. 311-317, 2003, p. 313, FIG. 2, and the like).
If a means for inhibition can be provided against virus replication targeting a nucleotide sequence conserved in the viruses belonging to the genus Mastrevirus among the geminiviruses, it is expected that, besides infection with WDV, infection with various plant viruses belonging to the genus Mastrevirus can be effectively controlled. Although the method disclosed in International Patent Publication WO2004/101798 and the like are known as a method for preparing a transformed plant having sustainable resistance against geminivirus, the approach thereof is completely different from that of the present invention.